Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3-L1 adipocytes

A protocol was developed in 3T3-L1 adipocytes that resulted in the specific desensitization of glycogen synthase activation by insulin. Cells were pretreated for 15 min with 100 nM insulin, and then recovered for 1.5 h in the absence of hormone. Subsequent basal and insulininduced phosphorylation of the insulin receptor, IRS-1, MAPK, Akt kinase, and GSK-3 were similar in control and pretreated cells. Additionally, enhanced glucose transport and incorporation into lipid in response to insulin were unaffected. However, pretreatment reduced insulin-stimulated glycogen synthesis by over 50%, due to a nearly complete inhibition of glycogen synthase activation. Removal of extracellular glucose during the recovery period blocked the increase in glycogen levels, and restored insulin-induced glycogen synthase activation. Furthermore, incubation of pretreated 3T3-L1 adipocytes with glycogenolytic agents reversed the desensitization event. Separation of cellular lysates on sucrose gradients revealed that glycogen synthase was primarily located in the dense pellet fraction, with lesser amounts in the lighter fractions. Insulin induced glycogen synthase translocation from the lighter to the denser glycogen-containing fractions. Interestingly, insulin preferentially activated translocated enzyme while having little effect on the majority of glycogen synthase activity in the pellet fraction. In insulin-pretreated cells, glycogen synthase did not return to the lighter fractions during recovery, and thus did not move in response to the second insulin exposure. These results suggest that, in 3T3-L1 adipocytes, the translocation of glycogen synthase may be an important step in the regulation of glycogen synthesis by insulin. Furthermore, intracellular glycogen levels can regulate glycogen synthase activation, potentially through modulation of enzymatic localization.

Section: Figmentioning

confidence: 98%

Specific Desensitization of Glycogen Synthase Activation by Insulin in 3T3-L1 Adipocytes

Jensen¹,

Crosson²,

Kartha³

et al. 2000

“…Previous work has clearly shown that treatment of 3T3-L1 adipocytes to high concentrations of insulin (Ն100 nM) leads to disconnect in insulin signaling (17)(18)(19)26). However, early insulin signaling has been documented to remain intact in 3T3-L1 adipocytes made insulin-resistant by lower insulin concentrations between 0.5 and 5.0 nM (20,21).…”

Section: Fig 2 Cellular Cortical F-actin and Plasma Membrane Pip 2 mentioning

confidence: 98%

“…For example, chronic exposure to pharmacological doses of insulin (Ն100 nM) have been shown by several laboratories (17)(18)(19) to markedly attenuate expression levels and/or activity states of the IR, IRS-1, PI3K, Akt, and GLUT4 proteins in 3T3-L1 adipocytes and, thus, produce a defect in the ability of the cell to respond to subsequent acute insulin stimulation with an increase in GLUT4 translocation and glucose transport. On the other hand, similar defects in the ability of insulin to acutely regulate the glucose transport system can be induced by physiological doses of insulin (Յ5 nM) that are not associated with disturbances in early insulin signaling (20,21).…”

mentioning

confidence: 99%

Protective Effect of Phosphatidylinositol 4,5-Bisphosphate against Cortical Filamentous Actin Loss and Insulin Resistance Induced by Sustained Exposure of 3T3-L1 Adipocytes to Insulin

Chen¹,

Raman²,

Bhonagiri³

et al. 2004

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (Factin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nM) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mM) and high (25 mM) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP 2 and found a noticeable loss of this lipid from the plasma membrane. Using a PIP 2 delivery system, we replenished plasma membrane PIP 2 in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulininduced insulin resistance involving defects in PIP 2 /actin regulation.

“…Likewise, IRS-1 was found to be down-regulated in adipose tissue from diabetic patients (28) and in 3T3-L1 adipocytes treated chronically with insulin (43).…”

Section: Desensitization Of Insulin Signaling Is Associated With a Pimentioning

confidence: 99%

Phosphoinositide 3-Kinase-mediated Reduction of Insulin Receptor Substrate-1/2 Protein Expression via Different Mechanisms Contributes to the Insulin-induced Desensitization of Its Signaling Pathways in L6 Muscle Cells

Pirola¹,

Bonnafous²,

Johnston³

et al. 2003

104

Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/ protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviraldriven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.